1. Field of the Invention
The present invention relates to an enhanced chemiluminescent reaction especially for use in a diagnostic assay, particularly immunoassay, and to a diagnostic kit for use in the assay. A chemiluminescent reaction is a chemical reaction which results in the emission of light. The luminescent emission is generally of sufficient duration to enable the light emitted to be detected or measured, and thereby to allow the detection or quantification of an analyte. The chemiluminescent reaction with which this invention is concerned is that between a 2,3-dihydro-1,4-phthalazinedione (DPD), especially luminol, with an oxidant, especially hydrogen peroxide, and a peroxidase enzyme, especially horseradish peroxidase, which catalyses the oxidation of the DPD by the oxidant. The oxidation is accompanied by emission of light.
2. Description of the Prior Art
Luminescent assays making use of the above-mentioned peroxidase-catalysed oxidation of a DPD include several types. It includes predominantly assays wherein horseradish peroxidase is conjugated to a ligand in order to label it and a luminescent reaction is used to detect or quantitate the label. This category includes ELISAs, competitive EIAs and nucleic acid hybridization assays, based on peroxidase labels. However, assays for measurement of free peroxidase, e.g. for analytical purposes, are also included.
A review of luminescent assays has been published by L. J. Kricka, Clinical Chemistry 37, 1472-1481 (1991).
The sensitivity of the peroxidase-catalysed chemiluminescent oxidation of DPDs can be enhanced by including an enhancer in the reaction. Such enhancers include a 6-hydroxybenzothiazole (European Patent 87959B), a phenol selected from a narrowly defined class (U.S. Pat. No. 4,598,044), an aromatic amine selected from a narrowly defined class (U.S. Pat. No. 4,729,950) or phenols substituted in ortho and/or para positions by imidazole or benzimidazole (European Patent 296752B), all owned by National Research Development Corporation. A further number of aromatic amine enhancers are disclosed in European Patent Application No. 219352A (Minnesota Mining & Manufacturing Co).
The present invention involves a new approach to increasing light output from chemiluminescent reactions and therefore further prior art will be discussed below after the "Summary of Invention" where it can be understood in context.